Project Summary While there are an increasing number of useful chemotherapy and radiological methods available for cancer therapy, the predominant treatment strategy remains surgical resection of the tumor. The greatest challenges for the surgeon are 1) precisely and rapidly locating the primary lesion and 2) clearly defining the margins between tumor and normal tissues. Failure in the form of recurrence after surgery usually results from incomplete removal of all cancer cells. There have been a number of recent advances in the use of optical contrast agents for fluorescence imaging guided surgery (FIGS). However, the only FDA approved optical agents are non-targeted dyes that are mainly used for perfusion imaging and in some cases have increased uptake in tumor tissues. A number of molecularly targeted optical contrast agents have recently advanced to clinical trials. In addition, there are a number of optical probes that exploit the activity of enzymes such as proteases that are activated in the tumor microenvironment. We have developed optical ?smart probes? that produce a signal only when acted upon by a class of proteases found in activated macrophages within the tumor microenvironment. While these probes have already been proven to be valuable agents for real time tumor imaging during surgery using the FDA approved da Vinci surgical system, they still suffer from relatively high background in some tissues and limited dynamic range of signal that depends on local probe concentrations. To address these limitations, we outline the design of a paradigm-shifting class of optical probes that require multiple tumor-specific enzyme signatures for activation, as well as a FRET imaging strategy to enhance both sensitivity and selectivity of probes. We will couple these probes with camera and image processing systems that will further enable their eventual clinical translation. This proposal is built around a large body of preliminary data resulting from an active collaboration between the Bogyo Lab and Intuitive Surgical Inc. These initial results have validated the key concept of using protease probes to image tumor margins during surgery using existing FDA approved surgical systems and serve as a strong foundation for the proposed studies aimed at enhancing the utility of existing optical imaging probes in the surgical workflow.